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Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization

Nature Chemical Biology volume 9, pages 428–436 (2013)Cite this article

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Abstract

RAF kinases have a prominent role in cancer. Their mode of activation is complex but critically requires dimerization of their kinase domains. Unexpectedly, several ATP-competitive RAF inhibitors were recently found to promote dimerization and transactivation of RAF kinases in a RAS-dependent manner and, as a result, undesirably stimulate RAS/ERK pathway–mediated cell growth. The mechanism by which these inhibitors induce RAF kinase domain dimerization remains unclear. Here we describe bioluminescence resonance energy transfer–based biosensors for the extended RAF family that enable the detection of RAF dimerization in living cells. Notably, we demonstrate the utility of these tools for profiling kinase inhibitors that selectively modulate RAF dimerization and for probing structural determinants of RAF dimerization in vivo. Our findings, which seem generalizable to other kinase families allosterically regulated by kinase domain dimerization, suggest a model whereby ATP-competitive inhibitors mediate RAF dimerization by stabilizing a rigid closed conformation of the kinase domain.

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Figure 1: Development of BRET-based RAF dimerization biosensors.
Figure 2: Profiling RAF inhibitors using RAF dimerization biosensors.
Figure 3: Development of a RAS-dependent CRAF-BRAF dimerization biosensor.
Figure 4: A high-throughput chemical screen using the CRAFKD–BRAFKD biosensor identifies new modulators of RAF dimerization.
Figure 5: Screening of a kinase inhibitor library reveals widespread off-target effects on RAF dimerization.
Figure 6: Probing the binding mode of RAF dimer inducers with BRAF mutant biosensors.

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Acknowledgements

We thank B. Breton and M. Audet for advice with the BRET2 system, D. Uehling and co-workers at the Ontario Institute for Cancer Research for GSK2606414, and the Institut de recherche en immunologie et en cancérologie (IRIC) high-throughput screening platform. IRIC is supported by the Canadian Center of Excellence in Commercialization and Research, the Canada Foundation for Innovation and by the Fonds de Recherche du Québec en Santé. H.L. is a recipient of Cancer Research Society and Canadian Institutes for Health Research (CIHR) Banting postdoctoral fellowships. N.T. is a recipient of the CIHR Canadian Graduate Scholarship. M.B., F.S. and M.T. hold Canada Research Chairs. This work was supported by operating funds from the Canadian Cancer Society to M.T. (018046) and from the CIHR to M.T. (MOP119443) and F.S. (MOP36399).

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  1. Hugo Lavoie and Neroshan Thevakumaran: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signalling, Université de Montréal, Montréal, Québec, Canada

    Hugo Lavoie, Gwenaëlle Gavory, Abbas Padeganeh, Sébastien Guiral, Jean Duchaine, Michel Bouvier & Marc Therrien

  2. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada

    Neroshan Thevakumaran, John J Li, Daniel Y L Mao & Frank Sicheri

  3. Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

    Neroshan Thevakumaran & Frank Sicheri

  4. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    John J Li & Frank Sicheri

  5. Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada

    Daniel Y L Mao

  6. Département de biochimie, Université de Montréal, Montréal, Québec, Canada

    Michel Bouvier

  7. Département de pathologie et biologie cellulaire, Université de Montréal, Montréal, Québec, Canada

    Marc Therrien

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Contributions

H.L., N.T., F.S. and M.T. designed the experiments and wrote the manuscript. M.B. contributed to the theoretical framework surrounding BRET assay development and participated in the analysis of the BRET data and in the revision of the manuscript. H.L., with assistance from G.G., A.P., S.G. and J.D., conducted cell-based BRET analyses, the high-throughput chemical screen and TR-FRET in vitro binding assays. N.T., with assistance from D.Y.L.M., J.J.L. and H.L., performed AUC and X-ray structure analyses.

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Correspondence to Frank Sicheri or Marc Therrien.

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Competing interests

H.L., F.S. and M.T. have filed a patent covering the BRET-based method described in this study for detecting RAF dimerization.

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Lavoie, H., Thevakumaran, N., Gavory, G. et al. Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization. Nat Chem Biol 9, 428–436 (2013). https://doi.org/10.1038/nchembio.1257

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